Despite great efforts in the lab and clinic, glioblastoma multiformi (GBM) has a poor prognosis. Current therapy consists of surgery followed by radiotherapy and chemotherapy with temozolomide (TMZ). Recent studies in several laboratories have demonstrated that GBM cultures in defined medium provide a better model than classical cell lines maintained with fetal bovine serum. As a result, we have placed a series of brain tumors into culture. We used these cultures to test the hypothesis that cancer stem cells (CSC) are relatively resistant to chemotherapy, using neurosphere formation as an assay for CSCs. To our surprise, the concentrations of chemotherapy drugs required to inhibit neurosphere formation are much lower than those required to inhibit bulk cell proliferation or to induce cell death. Further study demonstrated that the inhibition of neurosphere formation is due to reversible quiescence of CSCs, which facilitates DNA repair, lessens chemotoxicity and hence, decreases the efficacy of chemotherapy. We considered strategies to enhance the effects of chemotherapy. We were drawn to a recent discovery that inhibition of Notch signaling forces quiescent fibroblasts to become senescent. Notch signaling with a gamma secretase inhibitor (GSI) enhances TMZ-induced senescence. Based on these results, we hypothesize that TMZ therapy can be enhanced by suppression of the Notch pathway. Our overall objective is to develop TMZ+GSI as a novel therapy. Aim 1 is to determine the mechanism by which GSI enhances TMZ therapy. We predict that the GSI enhances TMZ therapy by inhibiting the Notch pathway and its downstream target, Hes1. Aim 2 is to assess the biological responses to TMZ+GSI, including senescence. We also will determine the roles of p53 and cyclin-dependent kinase inhibitors. Aim 3 is to optimize conditions for the TMZ+GSI treatment. We will compare several treatment schedules and carry out TMZ+GSI therapy in vivo in immunocompromised mice. These experiments will provide a sound foundation for moving this novel therapy to the clinic.